Functionalized α‑Helical Peptide Hydrogels for Neural TissueEngineeringNazia Mehrban,† Bangfu Zhu,‡ Francesco Tamagnini,§ Fraser I. Young,‡ Alexandra Wasmuth,†

Kieran L. Hudson,† Andrew R. Thomson,† Martin A. Birchall,⊥ Andrew D. Randall,§ Bing Song,*,‡and Derek N. Woolfson*,†,#,||

†School of Chemistry, University of Bristol, Bristol BS8 1TS, United Kingdom‡School of Dentistry, Cardiff University, Cardiff CF10 3XQ, United Kingdom§Medical School, University of Exeter, Exeter EX4 4PS, United Kingdom⊥University College London Ear Institute, London WC1X 8DA, United Kingdom#School of Biochemistry, University of Bristol, Bristol BS8 1TD, United Kingdom||BrisSynBio, University of Bristol, Bristol BS8 1TQ, United Kingdom

*S Supporting Information

ABSTRACT: Trauma to the central and peripheral nervous systems oftenlead to serious morbidity. Current surgical methods for repairing or replacingsuch damage have limitations. Tissue engineering offers a potentialalternative. Here we show that functionalized α-helical-peptide hydrogelscan be used to induce attachment, migration, proliferation and differentiationof murine embryonic neural stem cells (NSCs). Specifically, compared withundecorated gels, those functionalized with Arg-Gly-Asp-Ser (RGDS)peptides increase the proliferative activity of NSCs; promote their directionalmigration; induce differentiation, with increased expression of microtubule-associated protein-2, and a low expression of glial fibrillary acidic protein; andlead to the formation of larger neurospheres. Electrophysiological measure-ments from NSCs grown in RGDS-decorated gels indicate developmentalprogress toward mature neuron-like behavior. Our data indicate that thesefunctional peptide hydrogels may go some way toward overcoming thelimitations of current approaches to nerve-tissue repair.KEYWORDS: hydrogel, nerve tissue engineering, peptide, RGD peptide, self-assembly, stem cell

■ INTRODUCTIONTrauma to the central and peripheral nervous systems (CNS andPNS, respectively) can lead to severe disabilities, with injuriescausing progressive degeneration and affecting long-termfunctionality of the tissue. In vivo repair of nervous tissue iscomplicated by the slow migration of macrophages at the injurysite, and the consequent delay in the removal of glycoproteins inmyelin, among other components, which inhibit neural tissueregeneration.1−3 In addition, the lack of upregulation of adhesionmolecules and development of glial scarring at the site of theinjury culminate to create an environment hostile to functionalregeneration.4,5

Neural tissue complexity, donor-site morbidity, and limitedtissue availability in autologous tissue grafts result in loss of nervefunctionality that cannot be restored using surgical methodsalone; often resulting in the recruitment of surrounding nerves tocompensate for the damaged tissue. Nonetheless, in the absenceof functionally superior alternatives, autologous grafts remain thepreferred treatment.6,7 These limitations suggest a need foralternative methods for nerve-tissue repair.

To devise new approaches to neural-tissue repair, first we needto understand the surrounding chemical and physical niche. Thisis because the extracellular matrix (ECM) influences cellmorphology and function.8,9 Research in this area hassignificantly advanced from using allografts,10,11 xenografts,12−14

and decellularized tissue for neural repair to the development ofbespoke tissue-engineered products that match in vivoconditions more closely.15,16 To achieve this, both natural andsynthetic materials have been used to support the proliferationand differentiation of isolated neuronal cells, including neuralstem cells (NSCs). Natural scaffolds such as ECM proteinsfibronectin, laminin, and collagen have been explored exten-sively, but with varied degrees of success;17−20 ultimately, it hasbeen recognized that controlling the natural material’s chemicaland physical properties is difficult.21 Although synthetic materialssuch as poly(lactic-co-glycolic acid), poly(urethane), and

Received: January 30, 2015Accepted: April 28, 2015Published: April 28, 2015

Article

pubs.acs.org/journal/abseba

© 2015 American Chemical Society 431 DOI: 10.1021/acsbiomaterials.5b00051ACS Biomater. Sci. Eng. 2015, 1, 431−439

This is an open access article published under an ACS AuthorChoice License, which permitscopying and redistribution of the article or any adaptations for non-commercial purposes.

biodegradable glass offer tighter control over many suchproperties,22−26 their compatibility with basic cell functionssuch as growth, differentiation, and tissue formation isreduced.21,27

The natural progression is to combine favorable elements ofboth natural and synthetic systems to produce materials thatallow cellular attachment, migration, proliferation and differ-entiation, and for the diffusion of waste and nutrients in and outof the scaffolds. To this end, a number of materials have beenproduced, including some based on self-assembling peptides. Forexample, β-sheet-based systems have been explored using NSCswith some success,28,29 as have hybrid peptide−organic30 andpeptide−polymer systems.31,32 Here we present the functional-ization and use of hydrogelating self-assembling fibers(hSAFs).33 This α-helical coiled-coil peptide system has beenpurpose-designed to mimic some of the biochemical andmorphological properties of ECM. It forms porous hydrogels,which are suitable for the growth of model neural cells (FigureS1).34

To mimic the complexity of native ECM further, we needchemical cues to stimulate cells at different stages of tissuedevelopment: for example, fibronectin is expressed in earlyneuronal development to promote the growth of radial glialprocesses in the ventricular zone; and laminin is implicated in themigration of neurons.35−37 Furthermore, fibronectin is main-tained at low levels in the Schwann cell basement membrane ofadult peripheral nerves,38 but is rapidly upregulated followinginjury in both the PNS39 and CNS,40−43 and is recognized by atleast 10 different integrin receptors on the cell surface throughvarious combinations of α and β subunits.44 Thus, the expressionof guidance cues that stimulate and govern neuronal recruitmentand differentiation, including fibronectin, are complex and highlydynamic.Here, we focus on a cell-adhesion cue from fibronectin, namely

the Arg-Gly-Asp-Ser (RGDS) sequence,34 appending it to hSAFgels, and following its effects on primary NSC attachment,migration, proliferation, differentiation, and electrophysiology atthe early stages of cell development.

■ MATERIALS AND METHODSAn expanded version of all theMaterials andMethods used for this studyis available in the Supporting Information.Scaffold Formation. Peptides were synthesized using standard

solid-phase peptide synthesis protocols on a CEM “Liberty”microwave-assisted peptide synthesizer. Peptides were purified by reversed-phaseHPLC and their masses confirmed byMALDI-TOFmass spectrometry.Typically, hSAF gels were prepared by mixing separate 2 mM stocksolutions for each of the complementary peptides (hSAF-p1 or hSAF-p1(N3), plus hSAF-p2) in 20 mM MOPS (3-N-morpholino)-propanesulfonic acid, 5 mM sodium acetate, 1 mM EDTA) buffer atpH 7.4. This gave final solutions of 1 mM of each peptide. These wereleft on ice for 10 min followed by overnight incubation at 4 °C, resultingin gels. For decoration experiments hSAF-p1(N3) was substituted forhSAF-p1. After gel formation RGDS-decoration was performed byaddition of 2mM alk-RGDS and CuSO4 and ascorbic acid, each at 4 mMfinal concentration, at 4 °C overnight. As described previously,34 thisclick reaction proceeds to∼90%, presumably because some of the hSAF-p1(N3) peptides are protected through fiber/gel formation. Thus, weanticipate that∼90% of the hSAF-p1(N3) peptide, and 45% of the hSAFscaffold in total, should be decorated with RGDS units through thisprocedure. Gels were then washed with 10 mM EDTA (ethyl-enediaminetetraacetic acid) buffer, PBS (phosphate buffered saline),and NSC media. Half-moon gels were formed in 24-well cell-cultureplates using sterile glass coverslips as temporary separators for theundecorated hSAF and RGDS-decorated hSAF gels (Figure S1).34

Decoration of the hSAF-p1(N3) half of the gel was achieved as outlinedabove. Laminin scaffolds were formed by coating the tissue-cultureplates with poly-D-lysine for 1 h, washing with PBS and incubating withlaminin solution at 37 °C for 30 min. The plates were washed with PBSbefore use. All gels were made to thicknesses of ∼2 mm.

Murine Embryonic Neural Stem Cell (NSC) Isolation andCulture. Meninges were isolated from embryonic E12−E14 C57/Black6 murine cortices into ice-cold DMEM/F-12 media containingpenicillin−streptomcyin. After tissue digestion in accutase at 37 °C for10 min, the isolated cells were centrifuged, resuspended in NSC media,and incubated at 37 °C, 5% CO2. GFP-positive cells were producedaccording to a previously reported protocol45 and incubated at 37 °C,5% CO2. Media was replaced every 3 days and the cells passaged every 6days. After cell expansion, neurospheres were digested into single-cellsuspensions using accutase and resuspended in fresh NSC media beforebeing passed through a 40 μm cell strainer to remove remainingneurospheres. The cells were counted in the presence of Trypan Blue,seeded at a density of 0.5 × 104 in 96 wells and 1.0 × 104 in 24 wells, andincubated at 37 °C, 5% CO2.

Cell Studies. The proliferation rate of NSCs on hSAF and laminingels was evaluated by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) metabolic activity absorbance assay(Figure S3; protocol adapted from ref 46). For assessing cell migration,time-lapse images of NSC-seeded half-moon gels were recorded every10 min for 24 h. Migration was quantified as described previously.47 Forprotein expression and fluorescence microscopy cells were fixed withparaformaldehyde, their membranes permeabilized, nonspecific bindingblocked with bovine serum albumin at 22 °C for 30 min before rabbitpolyclonal anti-MAP2 and mouse monoclonal anti-GFAP primaryantibodies were added to the samples and left at 4 °C overnight. Thesamples were then washed with PBS-Triton X-100 before TRITC-conjugated (Alexa Fluor 594) goat antimouse and antirabbit secondaryantibody IgG was added and left at 22 °C for 1h. After further washingsteps, the samples were mounted using a DAPI (4′,6-diamidino-2-phenylindole)-containing medium and imaged on a Delta-visionmicroscope system using either a fluorescein isothiocyanate ortetramethylrhodamine isothiocyanate filter. Z-stacks were collated ona confocal laser scanning microscope to determine the depth of cellmigration into the gel. The number of processes produced by each cellwere counted using ImageJ software. Electrophysiological characteristicsof the cells were determined via patch-clamp tests (Figure S5).Recordings were performed after 7 and 14 days culture on gels bycontinuously perfusing the gels with Hank’s Balanced Salt Solutioncontaining 130 mM NaCl, 3 mM KCl, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-free acid, 1 mM MgCl2, 2 mMCaCl2, and 10 mM glucose, pH 7.3, 32 °C. Cells were visualized usinginfrared differential interference contrast microscopy combined withepi-fluorescence and patched using glass micropipettes filled 135mMK-gluconate, 5 mMNaCl, 10 mMK-HEPES, 200 μMEGTA, 300 μMNa-GTP, 4 mM Mg-ATP, 13.4 mM biocytin, pH 7.3. The liquid junctionpotential was corrected arithmetically. Following a whole-cell-recordingconfiguration, the pipet capacitance was neutralized and the seriesresistance compensated (10−80% correction). Voltage-clamp record-ings were made for the quantitative evaluation of voltage-gated currents.

Statistical Analysis. Data are presented in the format “mean ±standard error of the mean (SEM)”. Differences in mean values werecompared within groups and significant differences were determined byANOVA (analysis of variance) with posthoc Tukey−Kramer HSD(honestly significant difference) test. The significance level was set at p <0.05. For the electrophysiology experiments specifically, the maximalconductance values and the values for half the voltage that generated halfthe maximal conductance for K+ current activation were comparedbetween groups over time with two-way ANOVA. If an overall effect ofgel, time, or gel versus time was observed, a Bonferroni posthoc t test wasperformed. The same approach was applied to membrane passiveproperties between gels over time. The time of rise between 10 and 90%of the outward current peak (10−90 time of rise) at 14 days wascompared between gels with an unpaired Student’s t test.

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■ RESULTSExpression of Progenitor Characteristics, Proliferative

Activity, and Formation of Neurospheres. First, wecompared the response of NSCs to seeding on differentsubstrates; specifically, undecorated and RGDS-decoratedhSAF gels with laminin gels as a control (Figure 1). We followedthis using GFP-labeled nestin. This showed that NSCs expressedthe predifferentiation, intermediate-filament-protein marker48

on all three substrates over 14 days (Figure 1A−C and FigureS2), and therefore retained their ability to differentiate intospecific lineages. In addition, a standard MTT metabolic assayshowed that, although the majority of NSCs on the undecoratedhSAF gels survived the course of the study, the proliferativeactivity remained the same over this period (Figure 1D). Bycontrast, on the RGDS-decorated gels, this activity doubled overthe two-week time course, mirroring that seen for cells seeded oncontrol laminin gels (Figure 1D). Finally, the appearances of thecells on the three substrates were very different over time: by day14, those seeded on laminin were more spread out (Figure 1A);whereas, those on the hSAFs were clumped together (we refer tothese as “neurospheres”). Moreover, the neurospheres formedon the decorated hSAFs were much larger than those observedon the undecorated gels (Figure 1B, C).

Further analysis of the neurospheres showed that the cellsextended in the X-, Y-, and Z-directions creating 3D clustersembedded within the hSAF gels, compared with relatively flatcultured cells on the laminin substrate (Figure 2A−C, VideosS1−S3). For the former, the sizes of the neurospheres variedconsiderably between gel types with the cell layer on RGDS-decorated gels being much thicker than those on undecoratedgels (36 ± 8 μm and 20 ± 2 μm, respectively), which were boththicker than with the laminin substrate (9 ± 1 μm). Cells onRGDS-decorated gels also formed connections between theneurospheres (Figure 2D, Video S4), which were less apparenton the undecorated gels.

Directional Migration of Cells. We found that evenwithout added growth factors, the RGDS functionality promotedNSC migration. To follow this, we used time-lapse images andspider-plot analysis of cell migration on laminin as a controls, and“half-moon gels” in which undecorated and RGDS-decoratedhSAFs were prepared side-by-side in the same cell-culture well34

(Figure 3A−H). These revealed random migration of cells fromtheir starting positions on all three gels (Figure 3I−K, VideosS5−S7). However, analysis of the border of the undecorated andRGDS-decorated hSAF gels in the half-moon experiments clearlyindicated preferential migration of NSCs toward the decoratedside (Figure 3H, L, Video S8). There was little difference in the

Figure 1. Nestin expression and proliferation of NSCs on hSAF and laminin gels. (A−D) Single-cell suspension of GFP-nestin positive murine NSCs(green) seeded on (A) laminin, (B) undecorated hSAF, and (C) RGDS-decorated hSAF gels. Main panels, Day 14; insets, Day 0. (A−C) Day 14. (D)Proliferative activity of NSCs on laminin (red), undecorated hSAF (blue), and RGDS-decorated hSAF (green) gels over 14 days as measured by anMTT assay (n = 3 for each time point).

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displacement and directionality of the cells on laminin, and ondistinct regions of the undecorated, RGDS-decorated hSAFs,

(Figure 3M, N, first three bars). However, when presented withthe border of the two different hSAF chemistries, cells migrated

Figure 2. 3D neurosphere formation byNSCs in hSAF gels. 3D reconstruction of z-stack fluorescent images on (A) laminin, (B) undecorated hSAF, and(C) RGDS-decorated hSAF gels. (D) 3D reconstruction of z-stack fluorescent images of cells on RGDS-decorated gels showing neurosphereconnections. DAPI-stained cell nuclei (blue) and nestin expression (green). (A−D) Grid scales: 24.75 μm.

Figure 3.Migration of NSCs across half-moon hSAF, and laminin gels. (A−H) First and last frames of NSC migration captured over 24 h. (A) Bright-field images of NSCs on laminin, (B) undecorated hSAF, (C) RGDS-decorated hSAF gels, and (D) the border between an undecorated and RGDS-decorated hSAF half-moon gel. Blue arrowheads indicate the direction of cell migration. (E−H) Final frame bright-field images of NSCs on (E) laminin,(F) undecorated hSAF, (G) RGDS-decorated hSAF gels, and (H) the border between an undecorated and RGDS-decorated hSAF half-moon gel at 24 hpostseeding. Migration tracks (red) indicate the overall movement over this period; and the arrowheads indicate direction of cell migration. (I−K)Migration tracks of cells over 24 h on (I) laminin, (J) undecorated hSAF, (K) RGDS-decorated hSAF gels, and (L) the border between an undecoratedand RGDS-decorated hSAF half-moon gel represented as spider plots. Each line indicates a separate cell showing the start (center of the plot) and end(black dot) positions of each cell. (M)Displacement of cells over 24 h. (N)Directedness of cells over 24 h: migration in the y-direction (cos θ = 0), to theright (cos θ = +1), and to the left (cos θ = −1) are presented. n = 15 (laminin), 24 (undecorated hSAF), 48 (RGDS-decorated hSAF), and 12 (borderbetween undecorated and RGDS-decorated gels).

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toward the RGDS-modified side and traveled further (Figure3M, N, fourth bars). The cells at the border also appeared totravel faster toward that side (Figure S4).Stem Cell Differentiation. Cells seeded on all three gels

produced neurite-like processes by day 7 (Figure 4A−C). Thisindicated that the NSCs were adhering and differentiating withan elongated cell morphology in all cases. The undecorated gelsshowed the lowest percentage of cells with processes, whereasthose on RGDS-decorated and laminin gave similar levels(Figure 4J). Immunocytochemistry staining for microtubule-associated protein-2 (MAP2), a neural lineage specific marker,revealed it to be expressed by cells on all three gel types (Figure4D−F). The number of cells expressingMAP2 was lowest for theundecorated hSAF gel, with cells seeded on laminin and RGDS-decorated hSAF gels showing similar levels of expression (Figure4K). By contrast, very little glial fibrillary acidic protein (GFAP),which is associated with astrocytes or star-shaped glial cells, wasexpressed on any of the gels (Figure 4G−I, L).Cell Maturation and Electrophysiological Properties.

Patch-clamp recordings in voltage-clamp mode were used toevaluate the K+ currents in NSCs cultured in undecorated andRGDS-decorated hSAF gels. Starting from a holding potential of−90 mV, 30 ms step depolarizations were applied, incrementallyincreasing from −80 to +30 mV. When sufficiently largedepolarizations were used, clear outward K+ currents wereobserved in all cells. After 7 days of culture on either substrate,these outward currents were relatively slowly activating,essentially noninactivating, reflecting an immature phenotype

(Figure 5A). By day 14, however, the outward current waveformhad matured in both cases, developing clearly observable degreesof current inactivation and faster rates of activation (Figure 5B).Furthermore, by this time, the contribution of the inactivatingcurrent component was significantly larger in the RGDS-decorated gel (p < 0.001). This was seen in the inactivatingcomponent of current, which was larger than the noninactivatingcurrent remaining at the end of the 30 ms depolarizing stimulus.In contrast, the inactivating current component amounted toonly around 25% of the noninactivating current in theundecorated gel. In line with a more-mature K+-channelcomplement in the RGDS-decorated hSAF gels at 14 days, therate of current rise was faster in cells recorded on this substrate.Analysis of the peak amplitude of the outward current across a

range of test potentials from −80 to +30 mV was used todetermine the conductance activated at each voltage. This wasthen normalized to cell capacitance, an electrical parameterdirectly proportional to cell-surface area, yielding specificconductance versus voltage curves. At 7 days these curves werequite similar in both gels and did not exceed 2.5 nS/pF (Figure5C); the peak specific conductance was smaller for cells on theRGDS-decorated gel than the undecorated gel. At 14 days,however, the conductance density was clearly larger for theRGDS-decorated gel samples (Figure 5D). For these, the currentdensity grew almost 4-fold between day 7 and 14, whereas, therewas only a marginal and nonsignificant increase for theundecorated gels.

Figure 4.Differentiation of NSCs on hSAF and laminin gels. (A, D, G) laminin; (B, E, F) undecorated hSAF; (C, F, I) RGDS-decorated hSAF gels. (A−C) Phase-contrast images showing cell morphology at day 7. (D−F) Representative fluorescent images showNSC expression ofMAP2 (red) andDAPI-stained nuclei (blue) on all gels. (G−I) Representative fluorescent images show NSC expression of GFAP (red) and DAPI-stained nuclei (blue) on allgels. (J) Percentage of cells producing processes after 14 days in culture on laminin (red), undecorated hSAF (blue) and RGDS-decorated hSAF (green)gels. Percentage of cells expressing the neuronal differentiation markers (K) MAP2 and (L) GFAP. n = 3 for all experiments.

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■ DISCUSSIONWe have shown that it is possible to increase the attachment ofmurine NSCs to de novo designed peptide hydrogels (hSAFs)through the addition of a cell-adhesion motif, RGDS. This motifalso influences the proliferation and differentiation rates of NSCsover 14 days. Cells produced processes and expressed MAP2,with little GFAP. Analysis of the migratory behavior of the cellsindicated that, when given the option of undecorated and RGDS-decorated hSAF gels, NSCs preferentially migrated toward theRGDS-rich regions. RGDS decoration also influenced thefunctional electrophysiological properties. Although the cellsdid not fire action potentials, the RGDS-decoration promotedchanges associated with mature neurons.Furthermore, we have shown that the proliferation of NSCs

led to the formation of neurospheres extending into hSAF gels,with the largest neurospheres forming in the RGDS-decoratedgels. It is unlikely that the hSAF fibers are physically constrictingthe cells as observations made in the first day of cell cultureshowed cells freely migrating through the gels. Furthermore, cellsformed connections between neurospheres in RGDS-decoratedgels, indicating free migration. It may be that cell−cellinteractions encourage neurosphere formation in hSAF gels, orvice versa. It has been shown that neural precursor cells grown asneurospheres mainly express receptors that interact withfibronectin and vitronectin.49 Consistent with this, we did notobserve neurospheres with laminin as the substrate; although wedid observe these with both hSAF gel types. Such neurosphereformation may be more favorable for in vivo studies, where asmall wound site would require high cell proliferative activity inorder to provide a suitable environment for new tissue ingress.Further analysis of the predifferentiation migratory behavior of

the cells showed that when presented with both undecorated and

RGDS-decorated hSAF chemistries the cells actively migratedtoward the RGDS-decorated gels, and traveled further and fasterin the zone between the two gel types. Although RGDS isprimarily an adhesion sequence, in vivo studies have shown ahigher expression of fibronectin in brain tissue when neuronsmigrate toward the cortical plate region.36,50 Cell surfacereceptors bind to the RGDS sequences through focaladhesions.51 This attachment at the leading edge of the cellsencourages preferential movement toward RGDS-rich regions.52

Movement along traction points initiated by the focal adhesionscreates tension in the cell body and allows the cell to release anyweaker attachments at the rear, and effect propulsion toward theRGDS side.53 This is a repeating process of cytoskeletalreorganization, attachment, maturation and then disassemblyvia actomyosin contraction and so the cell is able tomigrate alongthe surface.54,55 We posit that this mechanism may be in play inour system. Furthermore, others show that at low adhesivity cellslack the traction needed to propel forward, while at highadhesivity the cells cannot rupture the bonds at their rear andtherefore do not propel forward.56 We suggest further that theundecorated and RGDS-decorated hSAF gels are analogous tothese low and high adhesivity regions, respectively. In turn, thiscould explain why cells at the border between the two gelchemistries move the furthest and fastest; that is compared withthose in the separated undecorated or RGDS-decorated zones,which behave more sluggishly and similarly.Ultimately, isolating which RGDS-integrin combination is

active at various stages in our system is not straightforward, andwould likely require an analysis of all possible combinations ofintegrin α and β subunits at the adhesome, i.e., the protein−protein interaction network at the integrin-ECM level.57

Furthermore, the “inside-out” signals governing the cell

Figure 5. Activation dynamics of NSC K+ currents on RGDS-decorated and undecorated hSAF gels at (A, C) 7 and (B, D) 14 days. (A, B) Outward K+

currents evoked by a +80 mV voltage step, normalized to the plateau and averaged on undecorated hSAF (black) and RGDS-decorated hSAF gel (red).(C, D) Analysis of the specific conductance recorded on undecorated hSAF (black) and RGDS-decorated hSAF gel (red) with different voltage stepintensities. The Boltzmann sigmoidal fit of the conductance-voltage curves at each time-point and for each gel is shown. The extrapolated maximalconductance and relative values for half the voltage that generates half the maximal conductance are reported. n = 12 (undecorated hSAF) and 14(RGDS-decorated hSAF) for day 7 and 5 (undecorated hSAF) and 10 (RGDS-decorated hSAF) for day 14.

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interactions with the ECM53 as well as the “outside-in” signalsaffecting the intracellular processes58 are complex and not easilyinterpreted, especially in the predifferentiation stage as the cellsmove. For example, we recognize that laminin is a keycomponent of extracellular matrix with many different iso-forms59,60 and that these also influence neural-cell attachment,differentiation and growth.61 Similarly, physical and rheologicaldifferences may exist between laminin and hSAFs, which couldinfluence cellular behavior. Nonetheless, the ability to recruitmigratory neural cells as well as promoting cell attachment whichwe have demonstrated, allows a high level of control that may beof use in directing cells toward wound sites where the wound bedwould otherwise be unfavorable for cell growth and tissuerepair.62

For postattachment cells, we found the expression of MAP2 tobe similar on RGDS-decorated hSAF and laminin gels. In vivo,MAP2 is expressed to stabilize microtubules before dendriteproduction and axonal maturity.63,64 Combined, these pointssuggest that cells on these gels are differentiating and may beapproaching maturation. However, NSC progenitor cells alsohave the ability to differentiate into radial glia, astrocytes andoligodendrocytes.65 Our studies show that very little GFAP wasexpressed by cells on any of the gels tested. GFAP expression inparticular would indicate the presence of reactive astrocytes (amajor component of glial scars), which are responsible forastrogliosis; that is, the synthesis of a supportive network to fillthe damaged tissue void and ultimately form a glial scar.Astrogliosis is also known to be one of the main reasons forneural, particularly axonal, regeneration failure.66 Therefore, thelack of GFAP in our systems indicates that the regenerativecapabilities of NSCs in hSAFs are being retained.Electrophysiological experiments allowed us to further

investigate how neuron-like these cells were. Neurophysiologicalchanges are expected as the NSCs differentiate, and areinfluenced by many factors.67−70 To the best of our knowledge,no such data has been recorded from de novo-designed gels.Whole-cell patch-clamp recordings showed large voltage-gatedK+ currents; ion channels that play a key role in shaping neuralactivity in vivo,71 on hSAF gels. The K+ current was highest onRGDS-decorated gels over 14 days and together with a negativeshift in the voltage dependence of activation and depolarizationsthat exhibited a rapidly inactivating current component. The dataindicate that cells on these gels are developing into matureneurons.72−74

■ CONCLUSIONIn summary, we have described how functionalized peptidehydrogels (RGDS-functionalized hSAFs) can be combined withneural stem cells (NSCs) to alter and control cellular attachmentand differentiation compared with unfuctionalized, control gels.Moreover, cells in competitive studies, migrate toward RGDS-rich regions of the gels. Controlling these behaviors will becrucial in recruiting cells to the wound site in vivo.In addition, with the RGDS-functionalized hSAFs, cells form

large neurospheres on the peptide gels, and show signs ofmaturation toward neurons. We recognize that to promotefurther maturation, and to mimic the complexity and continuallychanging environment of native neural tissue, other function-alities will have to be introduced into our system. Nonetheless,both of these responses, together with the altered cellularbehaviors, are encouraging for using RGDS-functionalized hSAFgels as a starting platform for neural-tissue development andengineering.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acsbiomater-ials.5b00051.

Figures S1−S5, descriptions of Videos S1−S8, andextended Materials and Methods (PDF)Video S1: Depth of NSC penetration into laminin gel(AVI)Video S2: Neurosphere formation and depth of NSCpenetration into undecorated hSAF gel (AVI)Video S3: Neurosphere formation and depth of NSCpenetration into RGDS-decorated hSAF gel (AVI)Video S4: Formation of connections between neuro-spheres in RGDS-decorated hSAF gel (AVI)Video S5: Migration and spider plots of NSCs overlaminin (AVI)Video S6: Migration and spider plots of NSCs overundecorated hSAF gel (AVI)Video S7:Migration and spider plots of NSCs over RGDS-decorated hSAF gel (AVI)Video S8: Migration and spider plots of NSCs across thehSAF half-moon border (AVI)

■ AUTHOR INFORMATIONCorresponding Authors*E-mail: D.N.Woolfson@bristol.ac.uk. Fax: +44 (0)117 9298611).*E-mail: SongB3@cardiff.ac.uk.Author ContributionsN.M., B.S., and D.N.W. conceived the project. N.M., B.Z., F.T.,A.D.R., B.S., and D.N.W. designed the various experiments.N.M., A.W., K.L.H., and A.R.T. made the peptides. N.M., B.Z.,and F.I.Y. conducted the cell-culture experiments. NM and FTconducted the electrophysiology experiments. M.A.B., A.D.R.,B.S., and D.N.W. supervised the work and raised grant support.N.M., B.Z., F.T., F.I.Y., A.D.R., B.S., and D.N.W. wrote the paper.FundingThis work was supported by the Biotechnology and BiologicalSciences Research Council (H01716X, D.N.W. and M.A.B.); theEuropean Research Council (StG243261, BS; and ADG340764,D.N.W.); the Royal Society (UF051616, B.S.); the MedicalResearch Council (G1100623, A.D.R.); and the Engineering andPhysical Sciences Research Council (Bristol Chemical SynthesisCentre for Doctoral Training, EP/G036764/1, K.L.H.). D.N.W.holds a Royal Society Wolfson Research Merit Award.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe thank the D.N.W. and B.S. groups and Joe Beesley inparticular for useful discussions. We are grateful to the ChemistryElectron Microscopy Unit and the Wolfson Bioimaging Facilityat the University of Bristol for access to microscopes and advice.We thank Dr. Wei Cui from Imperial College London for thenestin enhancer EGFP construct.

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